Method to Improve Therapeutic Properties of Stem Cells

ABSTRACT

In one aspect, a method of preconditioning stem cells comprising exposing stem cells to low dose radiation (LDR) is provided. In another aspect, a population of preconditioned stem cells is provided, wherein the population of 5 preconditioned stem cells is obtained by exposing stem cells to LDR. Uses of the preconditioned stem cells are also provided. In other aspects, the stem cells are muscle stem cells.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 16/631,233filed on Jan. 15, 2020, which is a US National stage entry ofInternational Application No. PCT/CA2018/050883, which designated theUnited States and was filed on Jul. 20, 2018, published in English,which claims the benefit U.S. Provisional Application No. 62/534,905filed Jul. 20, 2017 and entitled Method to Improve TherapeuticProperties of Stem Cells, the entirety of which is incorporated hereinby reference.

FIELD

The present disclosure relates to methods for improving the regenerativeand therapeutic properties of stem cells and stem cells obtained usingthe disclosed methods. In particular, the disclosure relates to methodsfor improving the regenerative and therapeutic properties of stem cellsby exposing the cells to low dose radiation (LDR). The disclosure alsorelates to stem cells that have been exposed to LDR, and methods of usethereof.

BACKROUND

Skeletal muscle is the largest organ in human body and its long-termmaintenance depends on muscle stem cells, otherwise known as satellitecells. They represent the major population of resident stem cells inadult skeletal muscle [Sambasivan and Tajbakhsh, 2007]. These adult stemcells facilitate the postnatal growth, remodeling and repair of themuscle tissue. Upon muscle injury, routinely experienced by humansduring any physical activity, satellite cells relocate from the surfaceof muscle fibers to the area of injury across the muscle mass andproliferate extensively [Charge et al., 2004]. During this proliferationthey adopt one of two fates: 1) self-renewal and switching back tostand-by mode (can be called reserve cells), or 2) differentiation toform myoblasts that will fuse with damaged muscle fibers to repair themuscle. In fact, the regenerative capacity of skeletal muscle makes itone of the best-studied examples of mammalian tissue regeneration.Consequently, there is great promise for the treatment of musclediseases using satellite cells [Aziz et al., 2012].

Muscle diseases can be broadly divided into a) genetic (e.g. Duchennemuscle dystrophy, facioscapulohumeral muscular dystrophy), b) agingrelated (e.g. sarcopenia) and c) other disease related (e.g. cachexiaassociated with cancer, kidney failure, chronic obstructive pulmonarydisease or others). Collectively, the family of muscular dystrophiesrepresents a major medical issue and currently there is no cure for anyof them. Intensive efforts have been made towards developingregenerative therapeutic strategies that include the use of satellitecells [Bengal et al., 2017, Crist, 2017].

In the last decade, progress has been made in methods for isolation andmoderate enrichment of satellite cells. However, due to a number oflimitations these efforts have yet to translate into efficientmodalities to treat muscle diseases. For example, one of the limitationsis related to low yields of satellite cells that are not sufficient fortherapeutic transplantation purposes [Kuang and Rudnicki, 2008]. Ex vivoexpansion may improve the final yield; however, rounds of passagingtypically lead to loss of sternness, accumulation of DNA damage, higherimmunogenicity and other undesirable outcomes. Subsequently, these canresult in immunological rejection after transplantation and inefficientmuscle fiber formation/regeneration. In addition, muscle stem cells areunable to travel great distances from the point of injection, causingpoor integration into host muscles. It is not surprising that clinicaltrials using satellite cells or myoblasts for treating muscle diseasehave not yet been successful [Tedesco et al., 2010]. Alternativeapproaches include the use of pluripotent human embryonic stem cells(hES) or induced human pluripotent stem cells (ihPS) [Maffioletti etal., 2015]. Yet, lengthy protocols of directed satellite cell productionfrom hES or ihPS cells may still suffer from issues related to in vitrogrowth and expansion [Chal et al., 2015]. Therefore, there is a need forpreconditioning of in vitro or ex vivo maintained muscle stem cells thatwould help preserve and/or improve their muscle and stem cell identityand other qualities or functions that are key to successful therapy.

SUMMARY

Although the existence of a multipotent entity that gives rise to allcells in the body or a specific tissue lineage e.g., blood, waspostulated in 1909 by a Russian scientist Alexander Maximow, it was notuntil recently that the concept of an adult tissue stem cell (SC) wasrecognized and accepted1. We now know that almost all tissues in thebody harbour a small subset of multipotent, quiescent cells capable ofself-renewal, proliferation and differentiation into mature cellularsubtypes. These cells are activated upon tissue damage, mobilize, divideand differentiate to replace diseased, aging or damaged tissues. Withthe discovery of the unique properties of stem cells and theirregenerative capacity came the possibility of utilizing these cells fortherapeutic purposes.

While very promising, stem cell therapy is thwarted by the challenge ofobtaining SCs in numbers that are sufficient to support therapydevelopment. Stem cells constitute a very small percentage of all adultcells in a given tissue—on the order of 0.01-0.001%. Therefore, it isnecessary to expand stem cells in vitro using specialized mediaformulations. Unfortunately, this ex-vivo manipulation of stem cellsleads to their premature aging, loss of stemness and significantlydecreased functional and regenerative capacity4. As a result, the onlycurrently approved stem cell therapy in North America is hematopoieticstem cell transplantation, which does not require any ex-vivo stem cellexpansion. Thus, methods that delay stem cell aging and improve SCsfunctional and regenerative capacity following expansion in vitro arehighly desirable.

This disclosure describes improving the regenerative and therapeuticproperties of stem cells by exposing the cells to low dose radiation(LDR). In some examples, the methods described herein can be used tohelp retard the aging and age-related degradation of one or moreattributes and/or properties of the particular stem cell type. That is,aged irradiated stem cells may experience less age-related degradationof one or more target attributes when compared to young stem cells thanan aged, non-irradiated stem cell. For example, stem cells that areirradiated may exhibit slower declines in one or more of theirattributes, such as proliferation, differentiation, fusion index as thecells age, than non-irradiated stem cells of the same type.

In one example, the inventors have demonstrated that irradiated musclestem cells may exhibit less age-related degradation of one or moreattributes such as their efficiency to the capacity of myoblasts todifferentiate into muscle fibers is enhanced if the cultures are exposedto LDR. The inventors have also demonstrated that the techniquesdescribed herein may, in some instances and for some types of stemcells, help enhance a functional capacity of stem cells and/or may helpdelay at least some aspects of age-associated decline in function. Theinventors have also demonstrated that upon stimulated differentiationmarkers of myogenesis are increased in cultures of myobtasts that havebeen exposed to LDR compared to unirradiated controls.

In other examples, the inventors have demonstrated that irradiatedmesenchymal stem/stromal and progenitor cells (MSC/MSPC) may exhibitless age-related degradation of one or more attributes such asproliferation and/or differentiation, as compared to non-irradiated stemcells of the same type and of analogous age/condition.

In other examples, the inventors have demonstrated that irradiatedendothelial colony forming cells (ECFCs)/endothelial stem cells (ESCs)suffer less age-related degradation of one or more attributes such asproliferation and/or migration potential, as compared to non-Irradiatedstem cells of the same type and of analogous age/condition.

In accordance with one broad aspect of the teachings described herein, amethod of preconditioning stem cells may include exposing stem cells tolow dose radiation (LDR), which may help provide preconditioned stemcells. This may, for example, enhance the functional capacity of stemcells and may help delay age-associated decline in stem cell function ascompared to similar stem cells that are not irradiated.

The radiation may be ionizing radiation, and optionally may beγ-radiation or X-ray radiation.

The cells may be exposed to 1 to 500 mGy of radiation, and optionally 5to 200 mGy of radiation or 8 to 150 mGy of radiation.

The stem cells may be muscle stem cells.

The stem cells may be mesenchymal stem/stromal and progenitor cells.

The stem cells may be endothelial colony forming cells/endothelial stemand progenitor cells.

The stem cells may be hematopoietic stem and progenitor cells.

The stem cells may be human stem cells or optionally may be mouse stemcells.

The stem may be exposed to LDR in vitro or ex vivo.

The preconditioned muscle stem cells may in some instances showincreased differentiation into muscle fibers compared to muscle stemcells that have not been exposed to LDR.

The preconditioned muscle stem cells may in some instances have higherexpression of at least one marker selected from the group consisting ofmyogenin, MyH3, MyoD, TKS5 and TMEM8c compared to muscle stem cells thathave not been exposed to LDR.

The method may include the step of administering the preconditioned stemcells to a subject in need thereof.

The subject may be a human.

The subject may have a muscle disease.

A population of preconditioned stem cells may be provided, and may beobtained by exposing stem cells to low dose radiation (LDR).

The radiation may be ionizing radiation, and optionally may includeγ-radiation or X-ray radiation.

The cells may be exposed to from about 1 to about 500 mGy of radiation,and optionally from about 5 to about 200 mGy of radiation or from about8 to about 150 mGy of radiation. The stem cells may be muscle stemcells.

The stem cells may be mesenchymal stem/stromal and progenitor cells.

The stem cells may be endothelial colony forming cells/endothelialstem/progenitor cells.

The stem cells may be human stem cells or optionally may be mouse stemcells.

The preconditioned muscle stem cells may show increased differentiationinto muscle fibers compared to muscle stem cells that have not beenexposed to LDR.

The preconditioned muscle stem cells may have higher expression of atleast one marker selected from the group consisting of myogenin, MyH3,MyoD. TKS5 and TMEM8c compared to muscle stem cells that have not beenexposed to LDR.

In accordance with another broad aspect of the teachings describedherein, a pharmaceutical composition may include a cell population ofpreconditioned stem cells, including those described herein, and acarrier.

In accordance with another broad aspect of the teachings describedherein, a method of treating a muscle disease in a subject may includeadministering at least some preconditioned stem cells described hereinto a subject in need thereof. The preconditioned stem cells may bemuscle stem cells.

The preconditioned muscle stem cells described herein may be used fortreating a muscle disease in a subject in need thereof.

In accordance with another broad aspect of the teachings describedherein, a method of preconditioning stem cells may include the steps of:

-   -   a) providing a sample comprising a plurality of target stem        cells;    -   b) irradiating the target stem cells with a first dose of        radiation emitted from a radiation source during an irradiation        period to convert the target stem cells to irradiated,        preconditioned stem cells suitable for use in a subsequent        therapeutic treatment process.

Irradiating the target stem cells may reduce age-associated decline ofat least a first cellular function of each target stem cell.

The first cellular function may have an initial performance value andmay define an aged performance value at a threshold aging time. Thepreconditioned stem cells may have a treated performance value at thethreshold aging time that may be between the aged performance value andthe initial performance value, and may be higher in some instances.

The treated performance value may be closer to the initial performancevalue than the aged performance value.

The target stem cells may each include a second cellular function havinga second initial performance value and defining a second agedperformance value at a second threshold aging time. The preconditionedstem cells may have a second treated performance value at the secondthreshold aging time and it may be between the second aged performancevalue and the second initial performance value.

The second threshold aging time may be different than the firstthreshold aging time.

The second treated performance value may be closer to the second initialperformance value than the second aged performance value.

At least one of the threshold aging time and the second threshold agingtime may be determined by the completion of a threshold number of cellpassages.

The threshold number of cell passages may be greater than 4, and may bebetween 4 and 23.

At least one of the threshold aging time and the second threshold agingtime may be determined by a time elapsed in a cell culture.

The target stem cells may include muscle stem cells. The first cellularfunction may include cellular fusion, the initial performance value mayinclude an initial fusion index, the aged performance value may includean aged fusion index, and the treated performance value may include atreated fusion index.

The treated fusion index may be greater than the aged fusion index.

The treated fusion index may be at least twice the aged fusion index.

The treated fusion index may be more than 50%, and may be more than 60%and more than 70%.

The preconditioned stem cells may show increased differentiation intomuscle fibers compared to target stem cells that are not irradiated.

The preconditioned stem cells may have higher expression of at least onemarker selected from the group consisting of myogenin, MyH3, MyoD, TKS5and TMEM8c compared to target stem cells that are not irradiated.

The target stem cells may include mesenchymal stem cells. The firstcellular function may include proliferation, the initial performancevalue may include an initial doubling time, the aged performance valuemay include an aged doubling time, and the treated performance value mayinclude a treated doubling time.

The treated doubling time may be less than the aged doubling time.

The treated doubling time may be less than 50% of the aged doublingtime.

The treated doubling time may be less than three times the initialdoubling time.

The threshold number of cellular passages may be between 12 and 15.

The threshold number of cellular passages may be 14 or 15, andoptionally may be 15.

The target stem cells may include a second cellular function that ischondrogenic differentiation. A second initial performance value mayinclude an initial differentiation capacity, a second aged performancevalue may include an aged differentiation capacity at a second thresholdaging time, and a second treated performance value may include a treateddifferentiation capacity at the second threshold aging time.

The target stem cells may include mesenchymal stem cells. The firstcellular function may be chondrogenic differentiation, the initialperformance value may include an initial differentiation capacity, theaged performance value may include an aged differentiation capacity, andthe treated performance value may include a treated differentiationcapacity.

The treated differentiation capacity may be greater than the ageddifferentiation capacity.

The treated differentiation capacity may be greater than the initialdifferentiation capacity.

The treated differentiation capacity may be at least 60% of the initialdifferentiation capacity.

The treated differentiation capacity may be at least 150% of the ageddifferentiation capacity.

A difference between the treated differentiation capacity and theinitial differentiation capacity may be less than a difference betweenthe aged differentiation capacity and the initial differentiationcapacity.

The target stem cells may include endothelial colony forming cells. Thefirst cellular function may be proliferation, the initial performancevalue may include an initial doubling time, the aged performance valuemay include an aged doubling time, and the treated performance value mayinclude a treated doubling time.

The treated doubling time may be less than the aged doubling time.

The treated doubling time may be less than the aged doubling time.

The threshold aging time may be defined by a threshold number ofcellular passages that is between 5 and 8, 6 and 8, 7 or 8, andoptionally may be at least 5 or at least 8.

A second cellular function may be migration. A second initialperformance value may include an initial time to achieve a predeterminedconfluency, a second aged performance value may include an aged time toachieve the predetermined confluency at the second threshold time, and asecond treated performance value may include a treated time to achievethe predetermined confluency at the second threshold time.

The predetermined confluency may be at least 60%.

The treated time to achieve a predetermined confluency may be less thanthe aged time to achieve the predetermined confluency.

The treated time to achieve the predetermined confluency may be betweenabout 1.4 and 1.8 times the initial time to achieve the predeterminedconfluency.

The target stem cells may include endothelial colony forming cells. Thecellular function may include migration, the initial performance valuemay include an initial time to achieve a predetermined confluency, theaged performance value may include an aged time to achieve thepredetermined confluency, and a treated performance value may include atreated time to achieve the predetermined confluency.

The predetermined confluency may be at least 60%.

The treated time to achieve a predetermined confluency may be less thanthe aged time to achieve the predetermined confluency.

The treated time to achieve the predetermined confluency may be betweenabout 1.4 and 1.8 times the initial time to achieve the predeterminedconfluency.

The target stem cells may be human stem cells.

The target stem cells may be mouse stem cells.

The radiation may include ionizing radiation.

The radiation may include low linear energy transfer (LET) ionizingradiation.

The radiation may include at least one of γ-radiation and X-rayradiation, and optionally may be γ-radiation.

The first dose of radiation may include between about 1 and about 500mGy of radiation.

The first dose of radiation may include between about 2 and about 200mGy of radiation.

The first dose of radiation comp may include rises between about 2 andabout 200 mGy of radiation.

The first dose of radiation may include between about 10 and about 100mGy of radiation.

The first dose of radiation may be about 10 mGy.

The first dose of radiation is about 50 mGy.

The first dose of radiation may be about 100 mGy.

The target stem cells may be irradiated while in vitro within a subjectto be treated.

The target stem cells may be irradiated while ex vivo.

The method may include using the preconditioned stem cells in asubsequent therapeutic process.

The method may include administering the preconditioned stem cells to asubject in need thereof. The subject may be a human, and optionally mayhave a muscle disease.

In accordance with another broad aspect of the teachings describedherein, a population of preconditioned stem cells may be obtained byusing the methods described herein

The preconditioned stem cells may include muscle stem cells and may showincreased differentiation into muscle fibers when compared to targetstem cells that have not been exposed to LDR at a threshold aging time.

The preconditioned muscle stem cells may have higher expression of atleast one marker selected from the group consisting of myogenin, MyH3,MyoD, TKS5 and TMEM8c compared to muscle stem cells that have not beenexposed to LDR.

In accordance with another broad aspect of the teachings describedherein, a pharmaceutical composition may include preconditioned stemcells obtained by using the methods described herein in combination withany suitable carrier.

In accordance with another broad aspect of the teachings describedherein, a method of treating a muscle disease may include administeringpreconditioned stem cells is obtained by using the methods describedherein to a subject in need thereof.

In accordance with another broad aspect of the teachings describedherein, a use of the preconditioned stem cells created in accordancewith the methods described herein may be used for treating a muscledisease in a subject in need thereof.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will now be described in relation to the drawings inwhich:

FIG. 1 shows muscle stem cells as a model for muscle aging andregeneration. Multiple rounds of this cycle throughout the life spanrepresent muscle stem cell aging through exhausting regenerativepotential and loss of muscle memory. In particular, muscle stem cellsundergo several divisions that result in proliferative expansion. Afraction of the dividing stem cells undergoes the self-renewing processand becomes the reserve stem cells. Upon receiving external signals,stem cells differentiate and fuse to form muscle fibers with reservecells as stem cells [Yaffe and Saxel, 1997]. The reserve cells canundergo this cycle again. Multiple rounds of such cycling and stem cellpool depletion result in aging characteristics. This process can bemodeled in vitro using C2C12 cells.

FIG. 2 shows an experimental design. C2C12 myoblasts were exposed to LDR(0, 10 or 100 mGy) and maintained via consecutive cycles ofdifferentiation and isolation of reserve cells (as described in FIG. 1 )for 30, 60 and 90 days.

FIG. 3 shows that LDR exposure reverses the decline of myogenicpotential with time of culture. A: Immunostaining for muscle specificmyosin heavy chain (MHC) by anti-MHC antibody of cultures maintained indifferentiation medium for 4 days (magnification 40×). B: Quantificationof the fusion index in differentiated C2C12 cultures of various ageswith or without LDR. Values are means of three independent experiments.

FIG. 4 shows that LDR exposure restores the levels of myogenic proteinsand genes in muscle fibers formed by long-term cultures of C2C12myoblasts. A: Untreated control and irradiated (10 and 100 mGy) culturesof various ages, as well as young cells were incubated in 2% horse serumfor 72 hrs to form myotubes. Western blot analysis on whole cell lysatesshowed a reduction in Myogenin and Myh3 in the untreated control, butlevels partially recovered in the irradiated cells. B: Sixty day oldcultures of untreated control and irradiated cells (10 and 100 mGy), aswell as young cells were incubated in 2% horse serum for 48 h. RNA wasextracted and RT-qPCR analysis was carried out to quantify mRNA levelsof various gene markers of myogenic differentiation and fusion. Datashows a sharp reduction with time of culture in differentiation markersMyogenin and Myh3 and fusion genes TKS5 and TMEM8c (Myomaker) in theuntreated control, but mRNA levels partially recovered in the irradiatedcells. Values are means of three technical replicates of a singleexperiment+/−SD.

FIG. 5 shows global gene expression profiling in mouse muscle cellsusing Next Generation Sequencing. A. Schematic representation ofexperimental plan for RNAseq by NGS. Young mouse muscle cells acutelyirradiated for 10 and 100 mGy doses and aged in culture for 60 days anddifferentiated to form muscle fibers. Samples were subjected for RNAseqanalysis using NGS technique. B. Gene expression in 10 and 100 mGytreated cells were compared to untreated control cells using CuffdiffNGS analytical tool and differentially expressed genes were representedas a venn diagram.

FIGS. 6A and 6B are graphs showing the gene ontology analysis ondifferentially expressed genes from the venn diagram in FIG. 5B. Treatedcells showed higher expression of genes required for muscle fiberformation via myogenic pathways and processes, refer table 1.

FIG. 7 shows observed beneficial effects from experiments conducted onbiopsy derived human muscle stem cells. A: is a graphical representationof experimental plan, young human muscle stem cells were exposed to LDR(0, 10 or 100 mGy) and maintained in growth media for 14 days anddifferentiated to form muscle fibers for 3 days in differentiation mediacontaining 2% horse serum. B: Representative immunofluorescencemicroscopy images from treated and untreated stem cells derived musclefibers. Graphical representation shows 60-70% increase in fusion indexin treated stem cells compared to untreated control stem cells n=3experiments.

FIG. 8 shows delayed aging in irradiated MSPC cultures. A: Doubling timeincreased from 24.4 to 95.5 hrs for MSPCs as they aged in culture fromp4 to p15. B: p4 cell were irradiated at 10, 50 and 100 mGy and allowedto age to p14 and p15. Measurements of doubling time were performed andplotted relative to doubling time of p4 untreated controls. Irradiatedgroups were compared to untreated control at the same passage.*Asterisks denote significant changes, p<0.5.

FIG. 9 shows delayed aging in irradiated ECFC cultures. A: Doubling timeincreased from 20.0 to 71.9 hrs for ECFC clone 13 as the cells aged inculture from p4 to p8. B: p4 cells were irradiated at 10, 50 and 100 mGyand allowed to age to p8. Measurements of doubling time were performedand plotted relative to doubling time of p4 untreated controls.Irradiated groups were compared to untreated control at the samepassage. *Asterisks denote relatively significant changes, p<0.5.

FIG. 10 shows increased chondrogenic differentiation of irradiated agedMSPCs. Young, p4 cells were acutely irradiated at 10, 50 and 100 mGygamma rays and allowed to age to passages 5 and 15. p5 and p15Irradiated cells were then differentiated along chondrogenic lineage for14 days in chondrogenic differentiation medium. Resultant chondrocyticpellets were processed, sectioned and stained for aggrecanas describedin Materials and Methods. A: Total fluorescent intensity of the pelletsections was quantified using ImageJ's Raw Integrated Densitycalculations. All values were normalized to untreated p5 controls.“Asterisks denote statistically significant (p<0.05) changes. B:Representative stained chondrocyte sections derived from aged p15untreated (UT) and 10 mGy irradiated cell pellets. Experiments wereperformed in duplicate. White bar represents 400 um.

FIG. 11 shows increased migration of irradiated aged ECFCs. Datarepresents a scratch wound assay. A wound was created in the cellmonolayer. Cells were monitored for 24 hrs and their migration capacitywas estimated by the speed of wound closure. A: Confluency of the woundwas measured at 10 hrs post scratch for aged untreated and treated cellsand expressed relative to the confluency achieved by young (p4)untreated cells. B: The time to achieve 60% confluency was estimated foraged untreated and treated cells and expressed relative to theconfluency achieved by young (p4) untreated cells. *Asterisks denotesignificant changes, p<0.5.

DETAILED DESCRIPTION

Various apparatuses or processes will be described below to provide anexample of an embodiment of each claimed invention. No embodimentdescribed below limits any claimed invention and any claimed inventionmay cover processes or apparatuses that differ from those describedbelow. The claimed inventions are not limited to apparatuses orprocesses having all of the features of any one apparatus or processdescribed below or to features common to multiple or all of theapparatuses described below. It is possible that an apparatus or processdescribed below is not an embodiment of any claimed invention. Anyinvention disclosed in an apparatus or process described below that isnot claimed in this document may be the subject matter of anotherprotective instrument, for example, a continuing patent application, andthe applicants, inventors or owners do not intend to abandon, disclaimor dedicate to the public any such invention by its disclosure in thisdocument.

Use of stem cells, including for example human stem cells, for treatinga wide range of human disorders and diseases in the clinic has beenexpanding over the last decade, but there are some limitations on thesepast usages. For example, some limitations may relate to variousfunctional properties of stem cells, such as the ability to proliferateand differentiate efficiently and robustly, to resist cell death andother suppressive signals, which may generally decline as stem cells arebeing manipulated ex vivo or in vitro.

One challenge with some existing practices involving the use of stemcells for such therapeutic treatments can be the time delay or lagbetween the provision of the original, young stem cells and theirultimate use in a therapeutic treatment. During this time period, thestem cells can age, which may affect one or more functions of the cell.For example, one or more cell functions of a given stem cell mayexperience age-associated declines in its function. This can, in someinstances, result in the aged stem cells having degraded cell functionswhich may affect their usefulness and/or effectiveness in the subsequenttherapeutic treatment. As described herein, the inventors havediscovered a method of preconditioning stem cells, including irradiatinga group of untreated, target stem cells, that may help reduce at leastsome types of age-associated decline of some cell functions as thetreated stem cells age, while still leaving the stem cells alive andsuitable for use in therapeutic treatment(s). As used herein, the term“preconditioning” can refer to the exposure of stem cells to an agent orstimulus to improve the regenerative and/or functional properties of thecells. For example, stem cells may be preconditioned prior totherapeutic transplantation.

Some protocols have been developed to try and help improve thetherapeutic properties of given stem cells that are to be used in suchtreatments, collectively called preconditioning, have been suggested,including, for example, exposure to hypoxic conditions, growth factorsand conditioned medium from other cells. According to literature, thesemethods may have various degrees of improvements, depending on stem celltypes, disease and end-points measured.

However, some of these preconditioning techniques can have limits and/ordrawbacks. For example, there have been some indications thatpreconditioning stem cells with hypoxia may lead to relatively poorerdifferentiation. Exposure to growth factors may be associated withhigher cost of the treatments. Incubating stem cells with conditionedmedium may require additional culture of “unaffected” stem cells whichin many cases may not be accomplished or does not apply to therapyprinciple.

Low dose radiation is commonly viewed as an additional health riskfactor to the public. However, contrary to this common viewpoint, theinventors have discovered that some cells may benefit from exposure tolow dose radiation, and that such exposure may actually improve thedesirable, therapeutic properties of the target cells. In particular, asdescribed herein, the inventors have discovered that the low doseradiation as a method to improve existing therapy methods, as comparedto some known radiobiology research that focuses on relatively highdoses of radiation, and have demonstrated some improvements in thecharacteristics of stem cells exposed to low doses of radiation.

Based on this discovery, and as an alternative to such conventionalpreconditioning techniques, the inventors have discovered a method ofpreconditioning stem cells, for example cells intended for therapeutictransplantation into a patient-recipient or any other potential use, byexposing them to low doses of radiation, such as ionizing radiationemitted from a suitable radiation source. The inventors have discoveredthat such exposure has been found to help preserve (i.e. slow thedeterioration/degradation of) certain cell functions of a given stemcell or group of stem cells, such as cell proliferation, cell viability,tissue specific memory, differentiation potential and other functionalproperties that are related to efficacy of therapy or to other uses, andmay help reduce the degradation of such attributes or functions overtime as the stem cells age. This may help the aged, preconditioned stemcells exhibit attributes that are relatively closer to the attributes ofyoung stem cells for longer time periods than non-preconditioned (i.e.control) stem cells of the same type.

This type of preconditioning can, in some instances, be distinguishedfrom other types of modifying and/or attempting to enhance theproperties of stem cells beyond the standard, or control properties ofuntreated, young stem cells. For example, some of the techniquesdisclosed herein use the unmodified attributes of young stem cells as abaseline/reference and are intended to reduce the degree to which theattributes decline or deviate from these baseline values as the stemcells age, as compared to intending modify the stem cells so that theirattributes exceed the baseline functions of the non-treated, young stemcell control group.

That is, the disclosed methods may, for some cells and some functions,reduce the age-associated decline of at least a first cellular functionof each target stem cell. For example, a given cellular function mayhave an initial performance value that can be measured on a group ofyoung (i.e. non-aged) stem cells, and which can be considered to form abaseline or control value for the given function.

Allowing at least some of the target stem cells to age without beingtreated as described herein can be used to define an aged performancevalue for the untreated stem cells. This aged performance value can bemeasured at any suitable point during the aging process, and can bedetermined at a threshold aging time which can serve as reference timefor the purpose of comparing the characteristics of treated andnon-treated stem cells. This threshold aging time can be defined by thepassage of time (for example may be measure in seconds, minutes, hoursand days or the like), or may be defined by other characteristics, suchas the number of rounds of passaging or cell passages.

The stem cells that have been preconditioned using the methods describedherein can also be allowed to age until reaching the threshold agingtime, at which point a measurement of the relevant cell function canestablish a treated performance value. It has been discovered by theinventors that treating the cells in the manner described herein canreduce the degradation of at least some cell functions, which can beunderstood as meaning that the treated performance value remains closerto the control, initial performance value than the aged performancevalue of the untreated cells (e.g. the aged, treated cells havefunctionality closer to the original young cells than the aged,non-treated cells). That is, for most measured values, the treatedperformance that may be between the aged performance value and theinitial performance value. In some examples of the methods describedherein, the treated performance value may be closer to the initialperformance value than it is to the aged performance value.

While some aspects of the methods herein are described with reference toa single cell function and its associated performance values, some usesof the methods herein may help preserve the values of two or more cellfunctions within a given stem cell. In such cases, different,respective, performance values may be calculated for each cell functionbeing monitored/compared. The effects of the methods on the differentcell functions may be analogous (i.e. that the preconditioned cells tendto have better performance values than the untreated, aged controlcells), but may have different magnitudes, proportions, relations, etc.based on the nature of the cell function being measured/compared.

Helping to maintain the functionality of the preconditioned stem cellsmay allow them to be stored for a longer time period before beingutilized in a given therapeutic treatment. This may help accommodatetravel/delivery times and/or may allow a batch of stem cells to beprepared in advance of when they are required and to be kept on handuntil needed. Alternatively, if the treated stem cells are utilized onapproximately the same time scale as non-treated cells would have beenutilized, they may show exhibit enhance performance and/or efficacy ascompared to the non-treated stem cells. This may it also helps expandthe cells to relatively large(r) numbers that are suitable for therapyapplications. For example, some stem cell therapies may require aminimum number of stem cells to achieve relatively high therapeuticindex, and delay in aging may help facilitate the expansion of suchcells for longer times and to larger numbers.

As used herein, the term “stem cells” may refer to cells that candifferentiate into specialized cells and can self-renew (i.e., divide toproduce more stem cells). Various types of stem cells are well known inthe art, and are contemplated in the methods disclosed herein. Examplesof stem cells include, but are not limited to, muscle stem cells,mesenchymal stem/stromal and progenitor cells (also known as mesenchymalstem cells or mesenchymal stem and progenitor cells), hematopoietic stemand progenitor cells (also known as hematopoietic stem cells) and andendothelial colony forming cells (also known as endothelial stem andprogenitor cells.

For example, in accordance with one broad aspect of the teachingsdescribed herein, which may be used in isolation and/or in combinationwith any of the other suitable aspects described herein, the inventorshave shown that exposing C2C12 myoblasts to low dose radiation (LDR) mayhelp enhance muscle stem cell memory (i.e. Inhibit the age-relateddecline in muscle stem cell memory function) which may help improvetheir potential to differentiate into muscle fibers. For example,exposure to LDR may enhance retention of muscle stem cells ability todifferentiate and form muscle fibers during extended in vitro growth.This property may help contribute to the successful therapeuticapplication of muscle stem cells in regenerative medicine and may beinversely related to the length of cell culture and/or aging. Theinventors have also shown that markers of myogenic differentiation wereincreased in cultures of C2C12 myoblasts exposed to LDR compared tounirradiated controls.

Accordingly, this disclosure provides at least one example of a methodof preconditioning stem cells comprising exposing stem cells to low doseradiation (LDR), thereby providing preconditioned stem cells havingenhance therapeutic properties as compared to similarly aged, andnon-preconditioned stem cells.

The attributes and/or therapeutic properties that may be enhanced by LDRpreconditioning may include, in some examples, delayed aging of thepreconditioned stem cells (as compared to the cell functions ofanalogous, non-precondition stem cells) and a corresponding delay inaging-associated loss of proliferation. It is noted that this need notinclude an improvement in the proliferation of relatively young stemcells (i.e. the proliferation of young LDR preconditioned stem cells maynot exceed the proliferation of young, non-preconditioned stem cells).Other functional capacities that may be enhance by LDR preconditioningmay include, in some examples, enhanced retention of muscle stem cellsability to differentiate and form muscle fibers during extended in vitrogrowth.

In some embodiments of the teachings described herein, the stem cellsmay be muscle stem cells. As used herein, the term “muscle stem cell”refers to stem cells present in skeletal muscle tissue, which canself-renew and are capable of giving rise to skeletal muscle cells.Muscle stem cells are also referred to as satellite cells. These stemcells are activated in response to muscle injury to regenerate damagedmuscle tissue.

Alternatively, the stem cells to be LDR preconditioned may bemesenchymal stem/stromal and progenitor cells (MSC/MSPCs). MSCs aremultipotent stromal cells that can differentiate into a variety of celltypes. Including, but not limited to: adipocytes, chondrocytes andosteocytes.

In some embodiments, the stem cells to be LDR preconditioned may beendothelial colony forming cells or endothelial stem and progenitorcells. Endothelial colony forming cells may give rise to endothelialcells that line all blood vessels, inner chambers of the heart andlymphatic vessels

Optionally, stem cells to be LDR preconditioned may be hematopoieticstem and progenitor cells (HSCs). HSCs may be located in the bone marrowand give rise to all blood cell lineages and platelets.

While experimental data is provided herein for the irradiation of someexemplary types of stem cells, it is expected by the inventors thatother types of stem cells, such as cardiac stem cells, may exhibitanalogous age-delaying behaviors when preconditioned in accordance withthe techniques described herein, including via irradiation with LDR. Forexample, different stem cells tend to have similar biology and function,i.e., they are quiescent cells that reside in specialized niches andwait for the physiological signals to migrate, divide and differentiate.Since delayed aging effects were demonstrated for 3 different stem celltypes with a similar degree of improvement the inventors believe it isreasonable to believe that other stem cells may behave in a similarmanner.

As used herein, the term “low dose radiation” (LDR) refers to a dose oflow linear energy transfer (LET) ionizing radiation that is similar to,or just above, natural background levels of radiation. Radiation dosagesof less than 500 mGy are understood to be low dose radiation levels and,in accordance with the teachings described herein, low dose radiationmay be a dose of ionizing radiation of less than about 500, 400, 300,200, 150, 125, 110, 100, 75, 50, 25, 12 or 10 mGy. In the examplesdescribed herein, some particular radiation doses have been demonstratedas helping to inhibit age-related degradation of stem cells.

The low dose radiation described herein may be provided using anysuitable irradiation source that can emit low linear energy transfer(LET) ionizing radiation in the dosage ranges described herein. Gamma(γ)- and X-ray radiation are two examples of suitable LET radiation thatcan be used for preconditioning. As will be understood by a person ofskill in the art, LET is an amount of energy deposited into a substancetraversed per unit length, i.e. keV/um. Anything below about 10 keV/umcan be considered to be low LET for use with the methods describedherein (for example γ- and X-ray radiation).

Accordingly, the suitable types of ionizing radiation can includeoptionally X-ray or γ-radiation. In one embodiment of the methodsdescribed herein, the ionizing radiation may be γ-radiation, and inanother embodiment, may be X-ray radiation. In the described methods,the stem cells may be exposed to radiation in doses of between about 1and about 500 mGy of radiation, and in some examples, may be betweenabout 2 and about 200 mGy, between about 10 and about 150 mGy ofradiation, between about 10 mGy and about 100 mGy. In somepreconditioning processes, the target stem cells may be exposed to about10 mGy, 50 mGy and/or about 100 mGy of ionizing radiation. In otherembodiments, the stem cells may be exposed to 8 to 12 mGy of radiation,optionally 10 mGy of radiation or 90 to 110 mGy of radiation, andoptionally 100 mGy.

Sources of γ-radiation for exposing stem cells to LDR include, but arenot limited to, ⁶⁰Co and ¹³⁷Cs, which can be used for medical purposes.In another embodiment, stem cells are exposed to LDR from an X-rayirradiator.

The stem cells may be irradiated (i.e. exposed to LDR) forpreconditioning through any suitable method, including those describedherein. The stem cells may optionally be exposed to LDR in vitro or exvivo.

In some embodiments of the methods described herein, the cells to be LDRpreconditioned may be in cell culture at the time of theirpreconditioning/exposure. For example, a population, or culture, of stemcells in a Petri dish or a test tube may be exposed to LDR.

Exposure times for irradiation, to achieve a desired level ofpreconditioning of a given stem cell can vary. The irradiation time fora given stem cell may be selected based on a variety of factorsincluding, for example, the desired dose of LDR, the method ofirradiation and available instrumentation. In some applications, thetime of exposure may vary between 1 s and 24 h. In one embodiment, thestem cells are exposed to LDR for 1 s 10 min.

In one embodiment, the aged stem cells that have been exposed to LDRhave improved (i.e. less degraded) properties compared to aged stemcells that have not been exposed to LDR. These improved properties mayinclude, but are not limited to, improved regenerative properties,increased or improved differentiation potential, increased viability,increased proliferation. Increased therapeutic efficacy, and increasedpreservation of stem cell properties. These properties may be assessedby any suitable method. Any of the attributes/properties may beincreased by at least 10, 25, 50, 75, 100, 200 or 300% compared to stemcells that have not been exposed to LDR.

For example, in embodiments where the stem cells are muscle stem cells,the aged muscle stem cells that have been exposed to LDR have shownincreased differentiation into muscle fibers as compared to aged musclestem cells that were not been exposed to LDR (that is, may have degradedless from the young cell control values than the untreated muscle stemcells).

Methods for assaying differentiation into muscle fibers are known in theart, and can involve quantifying the fraction of stem cells that becomeparts of newly formed muscle fibers.

For example, in one embodiment, a muscle fiber differentiation assay isused. In this example, a fusion index Is calculated using the formula:I_(f)=N_(fused)/N_(total)×100%, where N_(fused) is the number of nucleiwith myosin-positive cells (i.e., muscle fibers) and N_(total) is thetotal number of nuclei scored. A higher fusion index indicates increaseddifferentiation into muscle fibers. Accordingly, in one embodiment, themuscle stem cells that have been exposed to LDR have a higher fusionindex after at least 5, 10, 15, 30, 60 or 90 days of culture followingthe exposure, or within about 1 to about 100 days of culture followingirradiation, as compared to muscle stem cells that have not been exposedto LDR.

In other embodiments, stem cells that have been exposed to LDR may havehigher expression of at least one marker associated with celldifferentiation compared to stem cells that have not been exposed toLDR. For example, where the stem cells are muscle stem cells, the musclestem cells that have been exposed to LDR may have higher expression ofat least one marker associated with muscle cell differentiation comparedto muscle stem cells that have not been exposed to LDR. Markers ofmuscle differentiation are can include, but are not limited to,myogenin, MyH3, MyoD, TKS5 and TMEM8c. Accordingly, in one embodiment,the muscle stem cells that have been exposed to LDR have increased geneor protein expression of a marker of muscle differentiation, optionallymyogenin, MyH3, MyoD, TKS5 and/or TMEM8c, after at least 5, 10, 15, 30,60 or 90 days culture following the exposure to LDR compared to musclestem cells that have not been exposed to LDR. Many myogenic pathways maybe activated in the treated muscle stem cells confirmed by nextgeneration gene expression sequencing.

In some embodiments, the method can include obtaining or providing stemcells prior to exposing them to LDR. For example, the stem cells may beharvested from a tissue sample. As used herein, a “tissue sample” may beany sample of tissue that contains a stem cell. The tissue sample may beobtained from any mammal, including, but not limited to, humans andmice. In one embodiment, the tissue is muscle. As used herein, the term“harvesting cells” refers to isolating or extracting cells from a tissuesample such as muscle. Suitable methods of harvesting cells from tissuesamples are known in the art.

The stem cells used in the methods herein may be from any suitablesource, and may be generated from other cell types. For example,embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) maybe converted to muscle stem cells (Maffioleti et al., 2015 and Chal etal., 2015).

The stem cells may be grown and/or maintained in cell culture prior to,during and/or after exposure to LDR. As is commonly understood in theart, cell culture is the process by which cells are grown undercontrolled conditions, generally outside of their natural environment.Normally, cells in culture are maintained in culture media. As usedherein, the term “culture media” refers to media designed to support thegrowth of cells, in particular stem cells. Various culture media areknown in the art. In one embodiment, the culture media is a basal mediasuch as Dulbecco's modified Eagle's medium (DMEM), advanced DMEM,Biogro™, SkGM™, Ham's F10, Ham's F12, Iscove's modified Dulbecco'smedium, neurobasal medium, RPMI 1640 or MCDB120 medium. The medium maycontain serum or be serum-free. In one embodiment, muscle stem cells aregrown in DMEM with 10% FBS and differentiated to form muscle fibers inDMEM containing 2% Horse Serum, 5 μg/ml insulin and transferrin.

Optionally, the harvested stem cells are maintained in culture for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more passages, optionally 3passages, prior to exposure to LDR.

In other embodiments, the method further comprises administering thepreconditioned stem cells to a subject in need thereof.

Populations of Preconditioned Stem Cells

This disclosure also provides a cell population (for example, a cellculture), comprising stem cells that have been exposed to LDR. In oneembodiment, the cell population comprises preconditioned stem cellsobtained by the methods described herein. As used herein, the term“cell” refers both to a single cell and a plurality of cells. A“plurality of cells” may include a cell population.

Optionally, the preconditioned stem cells in the population can bepreconditioned muscle stem cells. Alternatively, the preconditioned stemcells in the population may be mesenchymal stem/stromal andprogenitor/cells or are endothelial colony forming cells/endothelialstem and progenitor cells. In yet another example, the preconditionedcells in the population may be hematopoietic stem and progenitor cells.The preconditioned stem cells may optionally be human stem cells.

Pharmaceutical Compositions

In accordance with another broad aspect of the teachings describedherein, pharmaceutical compositions may be created that include apreconditioned stem cell, created using the methods described herein, inas an active ingredient along with a suitable, pharmaceuticallyacceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Suitable carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field, which is incorporated herein by reference.Optional examples of such carriers or diluents include, but are notlimited to, water, saline, Ringer's solutions, dextrose solution, and 5%human serum albumin.

A pharmaceutical composition may be formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g. Intravenous, intradermal, subcutaneous, oral(e.g. inhalation), transdermal (i.e., topical), transmucosal, and rectaladministration.

The active ingredient may be prepared with a carrier that will protectit against rapid elimination from the body, such as asustained/controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

Optionally, oral or parenteral compositions may be formulated in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active ingredient calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms maybe dictated by and depend on the unique characteristics of the activeingredient (i.e. preconditioned stem cell) and the particulartherapeutic effect to be achieved, and the limitations inherent in theart of preparing such an active ingredient for the treatment ofindividuals.

The formulation may also contain more than one active ingredient asnecessary for the particular indication being treated, optionally thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the pharmaceutical composition cancomprise an agent that enhances its function. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

Uses of Preconditioned Stem Cells

Populations of preconditioned stem cells can be obtained according tothe methods described herein. Applications and uses of preconditionedstem cells may include, but are not limited to, preservation of stemcell properties during expansion, differentiation of stem cells intoparticular lineages (for example, differentiation of muscle stem cellsinto muscle fibers) and tissue regeneration. The differentiated lineagescan be used for both in vitro or in vivo purposes.

The preconditioned stem cells and pharmaceutical compositions describedherein may be useful for treating or preventing a disease or condition.Some examples of diseases or conditions that may be treated using sometype preconditioned stem cell(s), optionally those described herein, mayinclude muscular dystrophy, sarcopenia, Type 2 Diabetes, Septic Shock,multiple sclerosis, knee osteo arthritis, acute graft versus hostdisease, heart failure, Crohn s disease, acute myocardial infarction,acute myocardial infarction, pulmonary hypertension, and critical limbischemia.

Preferably, the disease or condition is a disease or condition known tobenefit from stem cell therapy. For example, the preconditioned musclestem cells and pharmaceutical compositions described herein may beuseful for treating or preventing a muscle disease or muscle condition.

The preconditioned muscle stem cells and pharmaceutical compositionsdescribed herein may optionally be used in a method for treating orpreventing a muscle disease or condition, the method comprisingadministering an effective amount of a preconditioned muscle stem cellor pharmaceutical composition disclosed herein to a subject in needthereof.

Optionally, an effective amount of a preconditioned muscle stem cell orpharmaceutical composition disclosed herein may be used for treating orpreventing a muscle disease or condition.

Optionally, the muscle disease or condition may be a genetic disease(for example, Duchenne muscle dystrophy or facioscapulohumeral musculardystrophy). In another embodiment, the muscle disease or condition is anaging-related muscle disease (for example, sarcopenia). In anotherembodiment, the muscle disease or condition is a muscle injury. In yetanother embodiment, the muscle disease or condition is non-genetic oraging related (for example, cachexia associated with cancer, kidneyfailure, chronic obstructive pulmonary disease or other).

As used herein, the term “subject” may include any suitable members ofthe animal kingdom, including, for example, mammals and in particular ahuman being. For example, the subject may be a patient having a diseaseor condition, such as a muscle disease or condition.

The preconditioned stem cells may be autologous stem cells (i.e., stemcells originating from the subject), or may be allogeneic stem cells(i.e., stem cells not originating from the subject).

An effective amount of a preconditioned stem cell or pharmaceuticalcomposition of the disclosure relates generally to the amount needed toachieve a therapeutic objective. Efficaciousness of treatment may bedetermined in association with any suitable method for diagnosing ortreating the disease. Alleviation of one or more symptoms of the diseaseindicates that the preconditioned stem cell or pharmaceuticalcomposition may confer a clinical benefit.

As used herein, “treating or preventing” includes, but is not limitedto, reversing, alleviating or inhibiting the progression of the diseaseor condition or symptoms or conditions associated with the disease orcondition. Preventing includes preventing occurrence of the disease orcondition or symptoms or conditions associated with the disease orcondition or preventing worsening of the severity of the disease orcondition or symptoms or conditions associated with the disease orcondition. Accordingly, “treating or preventing the disease orcondition” optionally includes the prophylactic treatment of a subjectto prevent or reduce the incidence or recurrence of the disease orcondition or symptoms or conditions associated with the disease orcondition.

Methods of administering stem cells to a subject are known in the art.For example, the preconditioned stem cells or compositions describedherein may be administered systemically or locally to a specific site ortissue of interest, in one embodiment, the preconditioned stem cells orcompositions described herein are injected into a subject. In anotherembodiment, preconditioned muscle stem cells are injected into muscle.

The preconditioned stem cells or compositions described herein may beused or administered in combination with another stem cell therapy orcell therapy. In this manner, the preconditioned stem cells orcompositions described herein may be used to augment the therapeuticcapacity of non-Irradiated cells.

The following non-limiting examples of methods of preconditioning someexamples of stem cells.

Example 1

Low doses of X-rays and γ-radiation have been shown to produce variousstimulatory effects at cellular and organismal levels, collectivelytermed radiation hormesis or radiation homeostasis [Calabrese, 2015;Calabrese, 2016; Baldwin and Grantham, 2015; Jolly and Meyer, 2009].Previous results demonstrated that low dose radiation (LDR), andspecifically low-dose γ-radiation, may delay the onset of tumorigenesisin vivo [Mitchel et al., 2003; Mitchel., 1999] and inhibit aging-relatedaccumulation of DNA lesions in vivo [Osipov et al., 2013]. Very littleis known about the effects of LDR on stem cells. The results of a fewstudies that examined the effects of LDR on stem cells are inconsistent.The inventors have discovered that the therapeutic properties of mouseand human muscle stem cells can be enhanced by preconditioning themuscle stem cells using LDR.

C2C12 mouse muscle myoblasts, a cell model commonly used in muscle stemcell biology, were used to study the effects of LDR on muscle stemcells. The results show that LDR exposure of C2C12 cells improves musclefiber formation by these cells and that loss of this capacity, routinelyobserved in a long-term culture, is partially reversed as a result ofLDR exposure of young cultures, as compared to unirradiated controls.

Methods C2C12 Mouse Muscle Stem Cells

These cells, also called myoblasts and originally derived from the hindlimb muscle of adult C3H mouse, were purchased from ATCC. C2C12myoblasts are efficiently fusing sub clone of C2 myoblasts and have beenused extensively as a model for muscle differentiation and muscle stemcell biology in tissue culture. For growth, cells were maintained inDulbecco's modified media (4.5 g/L glucose) supplemented with 10% fetalcalf serum, 4 mM L-Glutamine, and 1.5 g/L sodium bicarbonate while notallowing cell density to achieve greater than 80% confluency.

Human Biopsy Derived Muscle Stem Cell Culture

Muscle biopsy derived stem cells were purchased from a biotech companyLonza. To attain an exponentially growing culture, cells were maintainedin skeletal muscle basal medium supplemented with 20% FBS (fetal bovineserum), 4 mM L-Glutamine, Gentamycin, human EGF (epidermal growthfactor), Dexamethasone and 1.5 g/L sodium bicarbonate while not allowingcell density to achieve greater than 80% confluency.

Long-Term Culture

Muscle tissue regeneration, a continuous process that goes on throughoutan organism's life span, implies multiple rounds of successive changesbetween proliferation mode (myoblasts), differentiation andself-renewal, and stand-by or reserve mode (FIG. 1 ). To model this invitro, C2C12 myoblasts (proliferation mode) can be stimulated todifferentiate and self-renew, followed by a release of self-renewedstand-by cells, also called reserve cells, back into proliferation mode[Yaffe and Saxel, 1997]. Multiple rounds of these changes represent anin vitro model of exhausting myogenic potential of muscle stem cells(FIG. 1 ). This decline may mimic changes that occur in satellite cellsunder in vitro expansion conditions. Reserve cells exhibit manyclassical features of satellite cells, such as asymmetric stem cellOivision, higher expression of markers of specification such as Pax7 andpotential for differentiation. Functional properties of muscle stemcells undergoing multiple rounds of differentiation-proliferation woulddecline with the number of such rounds. Reserve cells were obtained fromdifferentiated muscle fiber culture by performing differential enzymaticdigestion.

Unlike C2C12 myoblasts described herein, freshly isolated muscle stemcells from muscle biopsy may have limited (10-12) doubling capacity.Therefore, instead of performing reserve cell isolation, muscle stemcells were maintained in growth media for 14 days and 3 days indifferentiation media.

Differentiation of C2C12 Muscle Cells into Muscle Fibers

Cultures grown to 80-90% confluency in growth medium were washed twicewith phosphate buffer saline and incubated in differentiation medium(DM: DMEM with 2% horse serum and antibiotics). DM was replaced everyday until the end-points were reached. Incubation in DM medium triggersthe process of differentiation, an essential part of which is the fusionof myoblasts into multinucleated muscle fibers. By 72 h ofdifferentiation approximately 50.70% of myoblasts are fused into musclefibers.

Differentiation of Human Muscle Stem Cells into Muscle Fibers

Cultures grown to 80-90% confluency in growth medium were washed twicewith phosphate buffer saline and incubated in differentiation medium(DM: DMEM-F12 1:1 with 2% horse serum and antibiotics).

Irradiation

In the present example, young C2C12 myoblasts were irradiated with 10 or100 mGy or sham-irradiated using a Gamma-Cell 200 device equipped with a⁶⁰Co source. Following irradiation, cells were maintained for up to 90days in culture as described in the “Long-term culture” sub-section(FIG. 2 ). At time-points 30, 60 and 90 days, cells were assayed forproliferation and differentiation end-points.

Young human biopsy derived muscle stem cells were irradiated with 10 or100 mGy or sham-irradiated using a Gamma-Cell 200 device equipped with a⁶Co source. Following irradiation, cells were maintained for up to 14days in culture as described in the “Long-term culture” sub-section. Atthe end of 14 days cells were shifted to differentiation media to obtainmuscle fibers and assayed for differentiation and fusion index.

Muscle Fiber Differentiation Assay

Cultures on glass coverslips, that have undergone differentiation forvarious periods of time, were fixed in 4% paraformaldehyde andimmunolabelled with a primary antd-MyH3 antibody (Myosin Heavy Chain)diluted in PBS/5% BSA solution overnight at 4° C. After washing, cellswere incubated with a secondary antibody conjugated with Alexa Fluor 488(Invitrogen-A11001). Prior to visualization, coverslips were mounted inmounting medium with DAPI (Vector Laboratories Inc. H-1500). Images weretaken using a Zeiss Epifluorescence Observer Z1 microscope under 40×magnification.

The fusion index was calculated using the formula:

I _(t) =N _(fused) /N _(total)×100%,

-   -   Where:    -   N_(fused) is the number of nuclei within myosin-positive cells        (muscle fibers) and    -   N_(total) is the total number of nuclei scored.

In total, 500 nuclei were scored per sample.

Western Blot

Whole cell lysates were prepared from differentiated C2C12 muscle fibersas follows. Cell pellets were resuspended in 1 pellet volume of modifiedbuffer C (20 mM Hopes pH 7.6, 1.5 mM MgCl₂, 650 mM KCl, benzonase (2.5units/10⁷ cells), 0.2 mM PMSF, 0.5 mM DTT, 5 mM β-glycerolphosphate, and1 mM sodium orthovanadate) and centrifuged for 30 min at 4° C.Homogenates were then diluted with 1 pellet volume of Buffer E (20 mMHopes pH 7.6, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM PMSF, 0.5 mM DTT, 5 mMβ-glycerolphosphate, and 1 mM sodium orthovanadate). Extracted proteinswere then recovered from the cells by centrifugation at 15,000×g for 30min at 4° C. Extracts were quantitated for protein concentration priorto SDS PAGE. Each quantified extract was loaded and resolved in agradient polyacrylamide gel and transferred to PVDF membrane for westernblot analysis. Antibodies were purchased from Santa Cruz Biotechnologies(MyoD, SC 304; Myogenin, SC 12732; Myh3, SC 53091 and Tubulin, SC23948).

Quantitative RT-PCR

Cell cultures were trypsinized and pellets were produced bycentrifugation. RNA was isolated from cultured cells using Qiazol(Qiagen). Attached cultures were washed once with cold PBS, residual PBSwas removed and cells were scraped into Qiazol (1 mL for 4×10⁶ cells and0.5 mL for 2×10⁶). The lysate was vortexed vigorously to shear genomicDNA and then stored at −70° C. until further processing. RNA waspurified following manufacturers instructions (Invitrogen). Briefly,samples were thawed at room temperature for 5 min, 200 μL chloroform permL of Qiazol was added, vortexed vigorously for 30 sec, and phases wereseparated by centrifugation at 12000 rpm. RNA in the aqueous phase wascarefully removed and processed using mi RNeasy mini kit (Qiagen cat#217004). Finally RNA was dissolved/eluted in nuclease free water(Eppendorf) and tested for quality and quantity using a Nanodropspectrophotometer and Experion. 2.5 μg of total RNA was used to generatecDNA (RT first strand Kit Qiagen cat #330404). cDNA was diluted 1:5 inH₂O and mixed with Sybr green master mix (2×SYBR Biotool) and 2.5 picomoles of primer. Analysis was done in triplicate using the 7900HTSequence Detection Systems cycler (BioRad) and the CFX manager software.Primers used in the quantitative RT-PCR were designed by Primer 3software and checked by BLAST (Basic Local Alignment Search Tool)analysis. Primer sequences used in this study are listed as follows,Myogenin Forward GGC TCA AGA AAG TGA ATG AGG C; Myogenin Reverse CGA TGGACG TAA GGG AGT GC; Myh3 Forward GCATAGCTGCACCTTTCCTC; Myh3 Reverse GGCCAT GTC CTC AAT CTT GT; TKS Forward CTT TGT GGG GAA GAT GCT CG: TKS5Reverse TCC TTC TGG CCA CCT TCA AT; TMEM8C Forward GCT CCT ATG CAA AGACTG GC; TMEM8C Reverse GGT CGA TCT CTG GGG TTC AT.

RNAseq

60 days old mouse muscle (C2C12) cell cultures were trypsinized andpellets were produced by centrifugation. RNA was isolated and purifiedfollowing manufacturer's instructions (invitrogen), as described inherein. Purified RNA was later subjected for quality control. 5 μg RNAused in library preparation and 75 base pair single end sequencingperformed as per standard illumina procedures for the Next Seq 500genome sequencer. RNAseq data were analyzed using the Bowtie, Tophat2Cuffdiff (CuffLinks v1) software suit. Sequencing reads were mapped toGRC m38 mouse genome assembly with HISAT2 v2.0.4, guided by GENCODE vm12gene expression model. Identification and quantification of differentlyexpressed genes were performed using Cuffdiff and data sets presented asa Venn diagram. Functional relevance of differentially expressed genesin the LDR (10 and 100 mGy) treatment group were interpreted using aGene Ontology (GO) pathway analysis software which specifically considerbiological processes (BP).

Statistical Analyses

All experiments except RNAseq were repeated three times from the stageof tissue culture initiation and LDR exposures, so they representbiological replicates. Mean values from the three replicates werecalculated. Statistical significance while comparing groups wasdetermined using the Student t-test at P<0.05.

Results

LDR Improves the Potential of C2C12 Cells to Differentiate into MuscleFibers

The potential of C2C12 myoblasts to form muscle fibers at varioustime-points during the long-term culture experiment and how thispotential may be affected by LDR was evaluated. The capacity to formmuscle fibers represents an important functional characteristic ofmuscle stem cells. It can be robustly measured experimentally bymaintaining myoblasts under differentiating growth conditions forseveral days and then quantifying the fraction of myoblasts that becameparts of newly formed muscle fibers. This is done by immunofluorescencemicroscopy, wherein muscle fibers are stained with MyH3 and the nucleiwithin the fibers are quantified relative to the total number of nuclei(FIG. 3 ). The resulting fraction of nuclei that are parts of musclefibers is called a fusion index.

It was found that in the control non-irradiated culture the fusion index(i.e. the control fusion index) drastically decreased in atime-dependent manner from 50% In young cells to less than 3% in 90-dayold cells (FIG. 3 ). If myoblasts were irradiated with 10 or 100 mGy atthe beginning of the culture experiment, the decline in the fusion indexwas not as pronounced as in the non-irradiated culture. The fusion indexwas higher in both 10 and 100 mGy irradiated cells (i.e. the treatedfusion index) compared to the control ones at 30, 60 and 90 days (FIG. 3). Exposure of young myoblasts to LDR delayed their functional declineresulting in 2-5 fold higher potential to form muscle fibers at advancedculture ages.

In this example, a treated fusion index was consistently greater thanthe aged fusion index (indicated as the UT bars in FIG. 3 ) at each ofthe threshold age measurement points (30, 60 and 90 days in thisexample), and was in some instances at least twice the aged fusionindex—and had values of at least 10%, 20% and 30% or more of the initialfusion index.

LDR improves the Potential of Human Muscle Stem Cells Differentiate toMuscle Fibers

In addition to mouse muscle myoblasts, experiments were performed onhuman muscle biopsy derived stem cells. It was observed that if themuscle stem cells were irradiated to form an irradiated culture, thatthe fusion index of the irradiated culture (i.e. the treated fusionindex) was greater than the fusion index of a control culture comprisinguntreated/unirradiated muscle stem cells (i.e. the control fusionindex). For example, referring to FIGS. 7A and 78 , if muscle stem cellswere irradiated with 10 or 100 mGy at the beginning of the experimentand age them in culture for 14 days followed differentiation for 3 days,as exemplified in FIG. 7A, irradiated culture showed increased fusionindex by a factor of about 2.5 fold compared to the untreated(unirradiated) control stem cells (as shown, for example in FIG. 7B). Inthe illustrated example, the treated fusion index was greater than theaged fusion index and was more than about 70% of the initial fusionindex (control) for both the 10mGy and 100mGy radiation doses.

Mechanisms of Longer Retention of Muscle Identity in LDR ExposedMyobtasts

To examine whether the enhanced muscle fiber formation in LDR-exposedmyoblast cultures of advanced ages was due to canonical muscledifferentiation pathway, several classical markers of terminaldifferentiation were quantified by western blot analysis in totalprotein extracts. These were myogenin, Myh3 (myosin heavy chain) andMyoD. Myogenin and MyH3 showed a considerable reduction upon long-termculture in the control group, whereas marked increases in these proteinlevels were found in irradiated cells (compare 60 and 90 days for 10 and100 mGy groups with the age-matched controls in FIG. 4A).

In this example, the preconditioned stem cells can achieve a higherconfluency after a given time (10 hrs in this case) than the untreatedcells (For the first sample max confluency increase observed was from67.9% (untreated) to 78.6% (treated). For the second sample max increasewas from 61.9% (untreated) to 77.2% for treated). That is, the treatedstem cells may take a shorter time to reach a given target confluency,and/or may reach a higher level of confluency in each time than thecomparison, untreated cells.

These observations were confirmed in an independent experiment in which60 day old cultures were differentiated and the expression of severalgenes, that are markers of differentiation and fusion, were assessed byquantitative RT-PCR (see, for example, FIG. 4B).

In addition, referring also to FIG. 5 , a comprehensive gene expressionanalysis using RNAseq of mouse muscle cells at day 60 strengthened thealready made observations. In the 10 and 100 mGy irradiated mouse musclecells, the markers of differentiation and fusion are improved whencompared to un-irradiated control, as shown, for example, in Table 1.

TABLE 1 Muscle Fibers Muscle Fibers [Myotubes] [Myotubes] 10 mGy 100 mGyFold Fold Gene Name Change p Value Change p Value Adam12 3.38 3.31E−122.14 3.05E−08 Actc1 2.76 2.18E−10 3.21 0 Adamtsl3 3.26 0 1.98 1.38E−11Tnnt2 0.88 0.000857 0.74 1.80E−05 Myog 1.86 1.39E−07 1.43 1.96E−09Tmem8c 1.37 0.000243 1.11 1.42E−05 ID3 2.008 9.07E−05 1.22 0.27756 Igf11.43 5.51E−06 1.39 1.70E−08 Igfbp3 2.56 1.13E−07 1.31 0.000422 Presentedfold changes of gene expression were obtained after normalizing withcontrol myotubes

The gene list obtained from data analysis suggests an improvement ofmuscle fiber formation. Majority of genes differentially expressed in 10mGy and 100 mGy have decisive functions in differentiation, muscletissue development, skeletal muscle fiber formation and musclecontraction, as shown in FIGS. 6A and 6B respectively.

Discussion

The retention of the capacity of C2C12 myoblasts normally declines withtime/cell age in culture and with the number of differentiation cycles.However, utilizing the methods described herein, the retention of thecapacity of C2C12 myoblasts has been enhanced by exposing the culturesto LDR. The observed 2.5 fold improvement of differentiation the humanmuscle stem cells, underscore at least some of the beneficial effects oflow dose radiation in the preconditioning of stem cells (FIG. 7 ).Noteworthy, limited doubling capacity of human muscle stem cells was abasis to restrict the described culture to 14-17 days rather than theextended 90 days in the immortal C2C12 mouse muscle cells.

Here, it was found that LDR (10 or 100 mGy) improved the differentiationcapacity of C2C12 cultures that were subjected to multiple rounds ofgrowth->differentiation->reserve cell fractionation. A decrease(16-fold) in the fusion index in the control unirradiated cellsassociated with the length of culture or with the number ofdifferentiation rounds was found. This decline was partially reversedwhen cells were exposed to LDR at day 7 of culture. Noteworthy, themagnitude of the improvement increased with time (from ˜50% at 30 daysto >300% at 90 days—FIG. 38 ), indicating that the effect of LDRexposure may be maintained for long periods of time.

However, next generation gene expression sequencing performed in 60 daysold mouse muscle cells not only showed induction of myogenic markers in10 and 100 mGy treated cells, but, referring to FIG. 5 , also snowedseveral classes of genes involved in muscle fiber formation, muscletissue development and muscle cell migration required for myogenicfusion and maturation, as shown also in FIGS. 6A and 6B.

It was further confirmed that classical markers of myogenicdifferentiation were increased in cultures that were subjected to LDRcompared to unirradiated controls, suggesting that the LDR may triggermolecular changes that ultimately converge on the canonical pathway ofmuscle fiber formation. Without being bound by theory, it is believedthat these changes could include mechanisms of retention of muscle stemcell identity. For example, regulation of the myogenicdifferentiation-specific gene expression by histone H3.3 variant may beone such mechanism [Ng and Gurdon, 20081. This possibility is in linewith reports showing that LDR may lead to epigenetic chromatinrearrangement (reviewed in (Miousse et al., 2017]).

The decline of the differentiation capacity in the control cultures wasnot accompanied by changes in proliferation rates of myoblasts. Noevidence was found that the proliferation rate was affected by LDRexposure. Without being bound by theory, the inventors believe this mayfurther suggest that the improvement of the differentiation capacity mayhave been due to qualitative changes in long-term cultures enablingenhanced retention of muscle identity.

The observed effect may have implications in stem cell-based therapiesof muscle disease. One current limitation of such therapeutic approachesis the necessity to expand muscle stem cells, either in ex vivo culturesof patient-derived muscle stem cells or in in vitro cultures of musclestem cells produced by directed differentiation from hES or ihPS cells.Preconditioning of cells in such cultures using LDR may help reinforceretention of their myogenic functional properties otherwise negativelyaffected by long-term culture conditions. This may help improve overalltherapeutic efficacy. It has been shown that LDR improves retention ofmuscle-specific identity in C2C12 mouse myoblasts subjected to multiplerounds of growth->differentiation->reserve cell isolation. Suchimprovement may find wide use in regenerative medicine, specifically inprospective stem cell-based therapies of various muscle diseases.

Example 2

In accordance with another broad aspect of the teaching describedherein, the effects of LDR on mesenchymal and endothelial stem cellswere investigated in accordance with the methods described herein.

Materials and Methods Cell Culture

In this example, umbilical cord blood (UCB) derived mesenchymalstromal/stem and progenitor cells (MSPCs) were obtained from AmericanType Culture Collection (ATCC, Manassas, VA, USA) and expanded in UCBmesenchymal stem cell (MSC) expansion medium (#PCS-500-030 andPCS-500-040, ATCC) following manufacturer's instructions. Endothelialcolony forming cells (ECFCs) were derived from fresh cord blood units(Canadian Blood Services, Ottawa, ON, CA) according to an approvedethical protocol (Veritas Independent Research Board). Cord blood wasprocessed using Ficoll Paque Plus density separation gradient (GEHealthcare Bio-Sciences AB, Uppsala, Sweden) to yield mononuclear cells(MNCs). MNCs were plated in CellBind coated 6-well plates (#3335,Corning, NY. USA) and supplemented with ECFC expansion medium (#CC-3162.Lonza Group Ltd., Basel, Switzerland). Medium was changed every 2-3days. All cells were allowed to adhere and expand in a standardhumidified C02 incubator at 37° C.

Once 80% confluency was reached for MSPC cultures and visible denseendothelial colonies emerged in ECFC cultures cells were passaged—p1.Individual ECFC colonies/clones were passaged separately using DOWCorning high vacuum Grease (DOW Corning Corporation, Midland, MI, USA)coated glass rings and seeded into separate wells in a 6-well plate at2.5×103 cells/cm2 and further expanded to p2 and in p100 to p3-4.Passaged MSPCs were re-plated into p100 plates at 3.0×103 cells/cm2 andexpanded to p2-4. At p4 all cells were acutely irradiated with 0, 10, 50and 100 mGy gamma rays using Gamma Cell 200 cell irradiator (AtomicEnergy of Canda Ltd., Chalk River, ON, CA) and allowed to age inculture. ECFC experiments utilized three different clones: 3-2, 3-3 and13 representing biological replicates. The experiments were performed,at least, in duplicate.

Functional Analyses

Aging was defined in this case as a gradual decline in proliferativecapacity (function) of cultured cells. A passage at which cell culturewas deemed “aged” differed for MSPCs and ECFC clones and was determinedto be p15 for MSPCs, p5 for ECFC clone 3-2, p8 for ECFC clone 13 and p11for ECFC clone 3-3. Growth curves for cell cultures were constructedbased on percent confluency measurements performed by Incucyteinstrument (Essen Bioscience, Inc., Ann Arbor, Michigan, USA). Incucytewas used to take images of cells with 4× and 10× objective every hour ofcell culture at every passage. Cell proliferation was measured asdoubling time in the linear portion of the cell growth curve, usuallybetween 20-80% confluency. The following formula was used for doublingtime calculations:

(t2˜t1)/(3.32×(log n2−log n1)), where

-   -   t2—final time point    -   t1—initial time point    -   n1—number of cells/confluency at t1    -   n2—number of cell/confluency at t2

Migration of ECFC cells was measured using scratch-wound assay and aCell Migration Kit (#4493, Essen Bioscience) according to manufacturer'sinstructions. Briefly, cells were seeded in a specialized 96-well plateat 90-100% confluency and allowed to adhere. All wells in the plate werescratched using a specially designed wound maker that allows forconsistent scratched to be performed to minimize well-well variation. Aplate was placed in Incucyte instrument and monitored for 24 hrs, everyhour an image was obtained using a 10× objective. The migration analysiswas based on two measurements: 1) amount of time it took to “heal” thewound to 60% confluency; and 2) the confluency of the wound at 10 hrspost scratch. All values were expressed relative to p4 non-irradiatedcells. This assay was performed with 5 replicates, on 2 clones: 3-2 and3-3 with aged cells representing p6 and p13, respectively.

Aged MSPCs were differentiated along chondrogenic lineage for 14 daysusing human MSC differentiation kit (#SC006, R&D System inc.,Minneapolis, MN, USA) following manufacturer's instructions. Briefly, p5and 15 cells were spun down to create a pellet and differentiated, withfresh medium changes performed every 2-3 days. Chondrocytic pellets weresectioned at 10 um using a cryostat (CM 3050, Leica Biosystems Inc.,Concord, ON, CA) and fluorescently stained with anti-aggrecan antibody.Aggrecan is a protein that is specifically produced in chondrocytes andact as a marker of chondrocytic differentiation. Images of pelletsections were captured with a 10× objective using Evos FL fluorescentmicroscope (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescentstaining was quantitated using ImageJ software (ImageJ v. 1.52b,National Institutes of Health, USA; http://imagej.nih.gov/ij) thatmeasures Raw Integrated intensity of all pixels in the image. All valueswere normalized to p5 untreated control and experiments were performedin duplicate.

Statistical Analysis

All values were plotted relative to untreated (UT) early passage (p4 orp5) cells. Treated aged groups were compared to untreated aged controlat the same passage and significance was determined using a paired,one-tailed student's t-test with p<0.5 representing significant changes.

Results and Discussion Delayed Aging of Low-Dose Irradiated Cells

To test the effects of LDR on aging of stem cells in culture, images ofuntreated and irradiated cell cultures were captured every hour forevery passage and percent confluency of the cell monolayer was measured.Using these values a growth curve was constructed and the doubling timeof cells was determined as described in Materials and Methods. FIG. 8Adepicts changes in doubling time of MSCPs as they age from p4 to p15with a relatively significant decrease in proliferation noted at p14-15.Similar observations were made for ECFCs with FIG. 9A representing theaging process of ECFC clone 13. Interestingly, when MSPCs and ECFCs areirradiated at early passage and allowed to age in culture, the agingprocess is delayed. FIGS. 8B and 9B demonstrate the delay in aging forMSPCs and ECFCS, respectively, as measured by decreased doubling timefor irradiated groups vs. untreated controls.

Increased Functional Capacity of MSPCs as Measured by IncreasedChondrogenic Differentiation

One of the defining features of mesenchymal stem/stromal cells is theirability to differentiate into cells of skeletal lineages such aschondrocytes. To test the effects of LDR on changes in functionalcapacity of MSPCs, passage 5 and 15 cells previously irradiated at p4were differentiated along chondrogenic lineage. Following 14 days ofdifferentiation chondrocyte pellets were sectioned and stained foraggrecan, amount of staining was quantified and is depicted in FIG. 10A.Differentiation capacity of p15 cells was expressed relative todifferentiation of young p5 MSPCs. There was an approximate 2× decreasein chondorgenic differentiation in untreated aged cells vs. untreatedyoung controls. However, upon LDR treatment aged cells maintained andeven improved their differentiation potential, e.g., 10 mGy condition.The representative images of chondrocyte pellet sections for aged UT andaged 10 mGy groups are shown in FIG. 10B.

Increased Functional Capacity of Low-Dose Irradiated ECFC Cells asMeasured by Enhanced Migration:

To evaluate the effects of LDR on the functional capacity of ECFCs, cellmigration assays were performed. One of the most important functionalattributes of endothelial stem cells is their ability to travel/migrateto the site of tissue damage and repair vascular networks. To test ECFCmigratory capacity scratch wound assays were performed with untreatedand irradiated aged ECFCs. All values were expressed relative to young(i.e. non-aged) untreated (i.e. non-irradiated) controls. Two separatemeasurements were performed as described in Materials and Methodssection. FIG. 11A depicts relative percent confluency of the wound at 10hrs after the scratch was made and FIG. 11B summarizes the relative timeit took for cells to reach 60% confluency within the wound. It isevident from both graphs that aged cells demonstrate decreased migrationin comparison to young cells, e.g., they are ˜70% as efficient atreaching confluency and taking ˜1.8 times longer to close the wound.However, aged irradiated cells while not performing as well as the youngcells still maintain majority of their migratory capability. FIG. 11thus demonstrates a delayed aging capacity of irradiated ECFCs when theyare expanded in culture as evident by the significant increase in theirmigratory capacity in comparison to untreated control.

While this invention has been described with reference to illustrativeembodiments and examples, the description is not intended to beconstrued in a limiting sense. Thus, various modifications of theillustrative embodiments, as well as other embodiments of the invention,will be apparent to persons skilled in the art upon reference to thisdescription. It is therefore contemplated that the appended claims willcover any such modifications or embodiments.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

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1-72. (canceled)
 73. A population of preconditioned muscle stem cells,wherein the population of preconditioned muscle stem cells is obtainedby using a method of preconditioning muscle stem cells, the methodcomprising: a. providing a sample comprising a plurality of targetmuscle stem cells; b. irradiating the target muscle stem cells with alow dose of radiation comprising between 10 and 100 mGy of radiationemitted from a radiation source during an irradiation period to convertthe target muscle stem cells to irradiated, preconditioned muscle stemcells suitable for use in a subsequent therapeutic treatment process;wherein irradiating the target muscle stem cells reduces age-associateddecline of at least a first cellular function of each target muscle stemcell.
 74. The population of claim 73, wherein the first cellularfunction has an initial performance value and defines an agedperformance value at a threshold aging time, and wherein thepreconditioned stem cells have a treated performance value at thethreshold aging time that is between the aged performance value and theinitial performance value.
 75. The population of claim 74, wherein thefirst cellular function is cellular fusion, the initial performancevalue comprises an initial fusion index, the aged performance valuecomprises an aged fusion index, and the treated performance valuecomprises a treated fusion index, wherein the treated fusion index isgreater than the aged fusion index.
 76. The population of claim 75,wherein the treated fusion index is at least twice the aged fusionindex.
 77. The population of claim 75, wherein the preconditioned musclestem cells show increased differentiation into muscle fibers compared totarget muscle stem cells that are not irradiated.
 78. The population ofclaim 75, wherein the preconditioned muscle stem cells have higherexpression of at least one marker selected from the group consisting ofmyogenin, MyH3, MyoD, TKS5 and TMEM8c compared to target muscle stemcells that are not irradiated.
 79. The population of claim 73, whereinthe radiation comprises low linear energy transfer (LET) ionizingradiation and comprises 7-radiation.
 80. The population of claim 73,wherein the dose of radiation is about 10 mGy.
 81. The population ofclaim 73, wherein first dose of radiation is 50 mGy.
 82. The populationof claim 73, wherein first dose of radiation is 100 mGy.
 83. Thepopulation of claim 73, wherein the population is for use in asubsequent therapeutic process comprising administering the populationto a subject in need thereof.
 84. The population of claim 83, whereinthe subject is a human.
 85. The population of claim 83, wherein thesubject has a muscle disease.
 86. The population of claim 73, whereinthe treated fusion index is more than 60%.
 87. The population of claim73, wherein the preconditioned stem cells comprise muscle stem cells andshow increased differentiation into muscle fibers when compared totarget stem cells that have not been exposed to LDR at a threshold agingtime.
 88. The population of claim 87, wherein the preconditioned musclestem cells have higher expression of at least one marker selected fromthe group consisting of myogenin, MyH3, MyoD, TKS5 and TMEM8c comparedto muscle stem cells that have not been exposed to LDR.
 89. A method oftreating a muscle disease comprising administering a population ofpreconditioned muscle stem cells of claim 73 to a subject in needthereof.
 90. A pharmaceutical composition comprising the population ofpreconditioned stem cells of claim 73 and a carrier.
 91. A use of thepopulation of preconditioned stem cells of claim 73 for treating amuscle disease in a subject in need thereof.
 92. The use of claim 91,wherein the subject is a human.